Use of immunogenic immunosuppressive and/or angiogenic proteins which have been rendered inactive, process for their preparation and pharmaceutical or vaccinal uses

ABSTRACT

The invention concerns the use of a protein derived from cancer cells, virus-infected cells or immune-defence cells or a fragment of said protein, characterized in that said protein is initially an immunosuppressive and/or angiogenic protein with local activity and said properties are inactivated by at least 70%, through a physical and/or chemical treatment, by genetic recombination or by adjuvant conditioning, said treatment preserving its property of being identified by antibodies directed afainst said protein, and preserving sufficient immunogenic properties for generating antibodies neutralizing or blocking said native protein, to obtain a medicine for use as local anti-immnumosuppression and/or anti-angiogenic agent as an anticancer agent. The invention also concerns the resulting immunogenic compounds, their preparation method and their uses.

The present application is a divisional of application Ser. No.09/743,700 filed on Jan. 16, 2001, which is a 371 national stageapplication of PCT/FR99/01423 filed Jun. 15, 1999, and the entirecontents of parent application Ser. No. 09/743,700 and PCT/FR99/01423are herein incorporated by reference.

The present invention relates to the use of immunogenicimmunosuppressive and/or angiogenic proteins which have been renderedinactive, a process for their preparation and their pharmaceutical orvaccinal uses.

Active compounds to combat cancer, which is the major medical scourge ofour time, are constantly being sought. Numerous therapies have beendeveloped with varying success, since there is still a high mortality.These therapies were initially surgical excision for solid tumours,radiotherapy and chemotherapy. These therapies seem insufficient in thecase of several cancers, for which no clinical success indicating asignificant prolonging of the survival of the patients or their totalrecovery has been recorded.

Cancers are proliferations of cells which can subsequently spread in theorganism to form metastases. It is known that the immune system of anormal individual regularly eliminates budding cancer cells(immunosurveillance concept) and that the formation of a cancer isassociated 1) with escape from the local immunosurveillance system andthen with an advanced period of cancer, to systemic immunosuppression,and 2) with a proliferation of vascular endothelial cells ensuringsupply of nutrients to the tumour cells (neoangiogenesis).

The majority of anticancer agents (chemotherapy, radiotherapy) used todate combat replication of cancer cells. These agents do not target theparticular environment necessary for proliferation of cancer cells,characterized both by the absence of local activity (paracrine) ofantitumoral immunity cells (immune escape) and by the occurrence of anintratumoral vascularization (neoangiogenesis).

New therapy approaches have therefore recently been introduced. Some areaimed at stimulating the antitumor immune system by cell therapy or byactivation of genes which code for proteins which stimulate the immuneresponse (gene therapy), or by direct immunization against antigens,identified as specific or associated, of the tumour of the MAGE type(vaccination). Others are aimed at combating neoangiogenesis usingantimetabolites which destroy the endothelial cells (Judah Folkman). Inthis new context it is to be noted that De Bruijn et al. (Cancer Res.(1998) 58, pages 724–731) have described the use of the native proteinsE6 and E7 to induce a cytotoxic cell response (CTL) in mice to protectthem against implantation of tumour cells.

The Applicant has now surprisingly discovered, after a long period ofresearch, that immunosuppressive or angiogenic factors which have aparacrine action are induced by tumours. These factors, which aresoluble, can on the one hand locally prevent the cells of the immunesystem from acting effectively even if they are stimulated(immunosuppression), and on the other hand ensure nutrition of thecancer cells by activating the proliferation of endothelial cells(angiogenesis).

The phenomenon of escape from the cell immune defences of the host byinduction of their paralysis in situ is a strategy used by severalcancers and is necessary for their survival. Immunosuppression initiallyremains localized in the tumour, since the individual is still capableof defence against other attacks, such as infections. However, at alater stage this immunosuppression may spread and become generalized, asdemonstrated by the dissemination of metastases and the highvulnerability of cancer patients to infections, to say nothing of thedebilitating effects due to chemotherapy or radiotherapy. Summarizing,this escape from the control of the immune system is due to a paralysisof the immune system (immunosuppression) which prevents normalfunctioning. This immunosuppression involves paralysing factors whichare produced by the cancer cells or their environment. Local paralysisof cells of the immune system or immunosuppression thus represents amajor weapon of cancer cells which enables them to escape from theimmune system of the host. Proteins released by the infected cells thusact as true toxins on the surrounding immunity cells, disturb them andblock in situ (in a paracrine manner) the cells of the immune system,protecting the infected cells.

Malignant tumours are characterized by the presence of significantvascularization, which ensures an inflow of blood necessary fornutrition of the cancer cells. This vascularization is realized byactivation of the induced vascular endothelial cells in contact with thetumour cells (neoangiogenesis). The works of Judah Folkman have recentlydemonstrated that control of tumour neoangiogenesis could represent adecisive effective weapon against cancers (Folkman J., Semin. CancerBiol., 1992, 3 (2): 65–71).

In this physiopathological context, the Applicant has discovered, aftera long period of research, three proteins involved in localimmunosuppression at the level of the tumour in the case of ATL, cancerof the cervix and Kaposi's sarcoma: these are the protein Tax of HTLV-1,the protein E7 of the papilloma virus, and the protein Tat of HIV-1 inKaposi's sarcoma. In this last case, an immunosuppressive protein ofHHV-8 is also said to exist, which would explain how Kaposi's sarcomaalso occurs in subjects not infected by HIV-1. Tat has been implicatedin Kaposi's sarcoma, but without researchers identifying its role as agenerator of local immunosuppression promoting generation of Kaposi'ssarcomas. Interestingly, the Applicant has found that some of theseimmunosuppressive proteins, such as the protein Tat of HIV-1 and theprotein E7 of HPV (strains 16 and 18) also have activating effects onvascular endothelial cells.

The object of the present invention is to block, by means of specificantibodies, the soluble factors released by tumour cells or cellsinfected by a virus. “Soluble factors” are understood as meaning factors(in general proteins) synthesized by the cells and released into theextracellular medium either by active transportation or by passivediffusion. The abovementioned extracellular factors can act in situeither by inhibiting the immunity cell response, including activation ofcytolytic T lymphocytes (CTL), or by disturbing the cytokine network, orby satisfying the nutritional requirements of the tumour byneoangiogenesis. These extracellular factors can be of cell origin(cytokines) or, for cancers induced by viruses or viral diseases, viralproteins, chiefly regulatory proteins, present in the extracellularmedium.

The present invention describes in particular means of obtaining, in thecirculating medium, antibodies directed specifically againstdeleterious, for example immunosuppressive and/or angiogenic, factors,the said antibodies being capable of blocking these factors andneutralizing the effects on the immunity and/or endothelial cells. Thesespecific antibodies will be either induced by active immunization(vaccination) directed against the proteins, in particular the solublefactors identified beforehand, or administered passively (passiveimmunization). The circulating antibodies present in the extracellularmedium will be able to neutralize the undesirable effects by combiningwith these proteins.

The Applicant has identified such soluble factors in at least threevirus-induced cancers: Kaposi's sarcoma (HIV-1), cancer of the cervix(HPV) and ATL leukaemia (HTLV-1 and -2). These 3 factors, all of viralorigin, are, respectively, the protein Tat of HIV-1, the protein E7 ofHPV and the protein Tax of HTLV-1.

Several cancers are induced by viruses, such as the HIV-1 responsiblefor Kaposi's sarcoma and other cancers, the HPV at the origin of cancerof the cervix, and the HBV and EBV associated, respectively, withhepatoma and Burkitt's disease.

The AIDS (Acquired Immunodeficiency Syndrome) disease due to HIV-1 andcharacterized by a generalized cellular immunosuppression can manifestitself by Kaposi's sarcoma, vascular cancer or by other forms of cancer,including certain leukaemias or lymphomas. The cell immunosuppressionobserved with this disease which promotes the occurrence of thesecancers is induced by the regulatory protein Tat of HIV-1, which,although it does not belong to the actual structure of the virus, isreleased by the infected cells into the extracellular medium. In thisextracellular form, the protein Tat, acting as a true viral toxin,exerts an immunosuppressive effect on the neighbouring immunity cells(Zagury D. et al.; PNAS, 1998, 95: 3851–56). Furthermore, at the levelof Kaposi's sarcoma, it has been shown that the protein Tat incombination with inflammatory cytokines (IFNα, Il-1, TNFα) and withBFGF, promoted the neoangiogenesis which forms the tumour (Ensoli B. J.Virol. 1993, 67: 277–287).

In fact, because of its immunosuppressive and angiogenic properties, theprotein Tat present in the extracellular medium promotes in cases of theAIDS disease not only the development of Kaposi's sarcoma caused by thevirus HHV-8, but also that of other cancers, including lymphomas orleukaemias.

The epithelial cancer of the cervix is caused by certain strains of theHPV virus (strains 16 and 18). The cancer cells of this cancer expressonly 2 proteins of early appearance from this virus, the protein E6 andthe protein E7, which both have effects on the regulatory factors of thecell cycle of the cancer cell. In addition, the protein E7 present inthe extracellular medium explains the occurrence of low levels ofantibodies against E7 in patients. The extracellular protein E7, likethe extracellular protein Tat, can act locally as a toxin on the stromalcells (lymphoid cells or endothelial cells) of the tumour.

This protein E7 has in fact shown immunosuppressive and angiogenicproperties in experiments. The immunosuppression induced by the proteinE7 was characterized by inhibition of the proliferation of T cellsstimulated by PPD or tetanus toxoid, inhibition of the proliferation ofT cells stimulated by allogenic cells, and over-production of IFNα(immunosuppressive cytokine) by antigen-presenting cells (APC). Theangiogenic potency of the toxin E7 on cultures of endothelial cellsoriginating from the umbilical cord of neonates and pretreated with theprotein E7 was suggested by the following observations: formation ofseveral cell nests, visible by phase contrast; identification by FACS ofCAM markers (ICAM and VCAM) within endothelial cells and modification ofthe cytoskeletons of cells in culture observed by immunofluorescence andalteration of the expression of nitrogen monoxide synthase which can beinduced by endothelial cells in culture in the presence of the proteinE7. As will be seen more decisively in the examples, the angiogenicpotency of the toxin E7 can be demonstrated directly “in vitro” byactivation of vascular endothelial cells originating from cell lines orfresh cells of the umbilical cord of neonates induced by the protein E7of HPV (strain 16).

The induction of antibodies directed against extracellular viralfactors, such as the proteins Tat, E7 or Tax, as toxins on the lymphoidstromal or intratumoral endothelial cells, involves the preparation ofimmunogens biologically deprived of the deleterious effects of thenative protein. As a result of their properties, such immunogens or thespecific antibodies which they induce can combat the immunosuppressionand/or angiogenesis present within tumours and can thus be used asanticancer medicaments.

The present Application chiefly relates to the use:

-   -   of a new product obtained from a natural protein which will be        modified by any technique, such as by a chemical or physical        (including the galenical form) route or by genetic engineering,        such that its immunosuppressive properties are inactivated to        the extent of at least 70%, preferably to the extent of at least        90%, in particular to the extent of at least 95%, by a chemical        or physical treatment and/or by suitable genetic construction.    -   of an antibody against an immunopathogenic, in particular        immunosuppressive or angiogenic, protein which has a local        action and is induced by a cancer cell or a cell infected by a        virus, to obtain a medicament intended for use as a local        anti-immunosuppression agent and/or as an anti-angiogenic agent        having a local action. Since cancers can proliferate as a result        of the local immunosuppression described above and of        angiogenesis, the above products are used in part to obtain a        medicament intended for use as an anticancer agent.

The abovementioned natural protein is characterized in that it is aninitially immunosuppressive and/or angiogenic protein which has a localaction and is induced by cancer cells or by cells infected by a virus,or a fragment of these proteins.

The present Application thus relates to the use of a protein originatingfrom cancer cells, cells infected by a virus or immunity cells, or afragment of this protein, characterized in that this protein is aninitially immunosuppressive and/or angiogenic protein which has a localaction and in that these properties are rendered inactive to the extentof at least 70%, preferably to the extent of at least 90%, in particularto the extent of at least 95%, by a physical and/or chemical treatment,such as formolation, carboxamidation, maleimidation or oxidation bybubbling oxygen through, by genetic recombination or by an adjuvantmake-up, the said treatment preserving the property of being recognizedby antibodies directed against the said protein and preservingimmunogenic properties sufficient to generate antibodies whichneutralize or block the said native protein, to obtain a medicamentintended for use as a local anti-immunosuppression or anti-angiogenicagent, as an anticancer agent. “Anti-immunosuppression oranti-angiogenic agent” is understood as meaning that the agent can haveone, the other or both effects.

The proteins which have been rendered inactive of the present invention,sometimes called “inactivated proteins” or “toxoids” in the following,allow combating of cancers by an approach which is complementary tothose of the prior art and specific, aimed at the paralysis of theimmune system and/or the angiogenesis induced by extracellularsubstances produced locally in the environment of the cancer cells. Thisimmunity paralysis and/or angiogenesis constitutes a true protectivebarrier and/or a source of nutrition for the tumour.

The proteins which have been rendered inactive of the present inventionallow combating in the first instance of these immunosuppressive and/orangiogenic factors by formation of antibodies against these proteins,and in particular against these soluble factors, in order to allow theimmune system to act effectively and/or to block neoangiogenesis.

It is important to use the deleterious extracellular factors in aphysically, chemically and/or genetically modified (inactivated) andnon-native (or non-natural) form so that it no longer exerts its harmfuleffects (paracrine paralysis of the immune system or localangiogenesis).

The physical treatments can be carried out by means of heat, UVradiation, X-rays or contact with an atmosphere rich in O₂. Thesephysical treatments, which generate intramolecular modifications betweenchemical radicals (for example thiol groupings), can appropriatelychange the conformation of the molecule and inactivate it functionally,while preserving its immunogenic properties.

The chemical treatment can be carried out with the aid of a couplingagent, such as a dialdehyde, or a carrier protein activated bypretreatment with the aid of a dialdehyde, preferably glutaraldehyde.The chemical treatment can be carried out using a monoaldehyde, inparticular formaldehyde. Reference may be made to the disclosure ofWO-A-96/27389 in this respect.

The chemical treatment can be carried out, in particular, by otherprocesses, such as carboxymethylation. An example of thecarboxymethylation technique is illustrated below in the experimentalpart. The chemical treatment can also be carried out byN-ethylmaleimidation, with or without glutaraldehydation.

The immunogen can be inactivated as a result of a galenical presentationwithin an oily liquid, such as incomplete Freund's adjuvant, or which iscapable of modifying the non-covalent bonds (electrostatic forces, Vander Waals forces or hydrogen bonds) necessary for its toxic effects.

The genetic modifications can be obtained by genetic engineering usinginsertions, deletions or substitutions of radicals, operations which areintended to reduce or suppress the deleterious functional sites of thenatural molecule. The genetic mutants could optionally be subjected to acomplementary chemical and/or physical treatment. The above modifiedproteins can be prepared, for example, from a protein having a sequenceidentical or similar to a peptide sequence of an immunopathogenic, inparticular immunosuppressive or angiogenic, protein, such as the proteinTat of HIV-1, the protein E7 of the papilloma virus or the protein Taxof HTLV-1, or of a fragment of these proteins and can be obtained, forexample, by conventional peptide synthesis on a resin or by geneticengineering. All these processes are well-known in the prior art.

In order to verify that the native immunosuppressive and/or angiogenicprotein is recognized properly by antibodies directed against the saidmodified immunosuppressive protein or its modified fragment according tothe invention, the formation of antigen-antibody complexes can beverified, for example, immunologically by ELISA in the presence ofspecific antibodies, as will be seen below in the experimental part.

Under the preferred conditions of use, the immunogenic compoundoriginates from a native compound (protein or polypeptide fragment)treated with an aldehyde or carboxamidated or maleimidated.

In order to determine whether the immunogenic properties of the modifiedimmunosuppressive and/or angiogenic protein or of a fragment of thisprotein have been preserved sufficiently (that is to say whether it hasbeen inactivated but not denatured) to create antibodies which block theeffects of the said native protein, it is possible, for example, toimmunize mammals (rabbits, rats, mice) with the aid of an immunogeniccompound according to the invention and to verify that the antibodiesproduced neutralize the immunosuppressive or angiogenic activities ofthe protein, as will be seen for the protein Tat of HIV-1, the proteinE7 of the papilloma virus and the protein Tax of HTLV-1 in theexperimental part.

In order to determine whether the modified immunosuppressive protein orthe fragment has lost at least the desired proportion of itsimmunosuppressive properties, it is possible, for example, to study theeffect of the immunosuppressive protein on immunosuppression of humanperipheral blood mononuclear cells (PBMC).

The modified and immunogenic immunosuppressive or angiogenic proteinscan be derived from any one of the proteins, in particularimmunosuppressive proteins, which haves a local action and are inducedby tumours; reference is made in particular to the protein Tat of thevirus HIV-1, the protein E7 of the papilloma virus or the protein Tax ofthe virus HTLV-1. Reference is also made to the mannan-dependent lectinproduced by activated immunity cells.

“Derived” or “to derive” from an immunopathogenic, in particularimmunosuppressive or angiogenic, protein which has a local action as isproduced by cancer cells or cells infected by a virus or produced byimmunity cells is understood as meaning that the immunogenic compoundcan be made up of all or a fragment of the immunopathogenic, inparticular immunosuppressive or angiogenic, starting protein.

It can include one or more modifications in the amino acids of thisprotein or fragment, such as deletions, substitutions, additions orfunctionalizations, such as acylation of amino acids, to the extent thatthese modifications remain within the context defined above (absence oftoxicity, immunological characteristics). For example, the replacementof a leucine radical by an isoleucine residue in general does not modifysuch properties; the modifications must generally affect less than 40%of the amino acids, preferably less than 20% and more particularly lessthan 10% of the immunopathogenic, in particular immunosuppressive orangiogenic, protein. It is important that the protein or modifiedfragment is not denatured, as can be effected, for example, by aphysical treatment, such as heat, in order to preserve itsconformational sites, so that the antibodies induced by the modifiedderivatives are active with respect to the native protein.

Under preferred conditions, the immunogenic compounds of the inventioncomprise at least 50% of the entirety or of a segment of theimmunopathogenic, in particular immunosuppressive or angiogenic,protein, preferably at least 70%, in particular at least 90%, and moreparticularly the entirety or virtually the entirety of the saidimmunosuppressive protein or of the angiogenic protein.

Generally, as regards the modifications, homology or similarity betweenthe modified immunogen and the native immunosuppressive protein or partthereof, as well as the dimensions of the immunogenic compound, andfurthermore the methods of use or the coupling of the immunogeniccompound according to the invention to an immunogenic protein, such asthe tetanus toxoid, reference may be made in particular to WO-A-86/06414 or to EP-A-0.220.273 or to PCT/US.86/00831, which are equivalents,the disclosure of which is incorporated here by reference.

The present invention also relates to an immunogenic compound,characterized in that it is an initially immunosuppressive and/orangiogenic protein which has a local action and originates from cancercells, cells infected by a virus or immunity cells, or a fragment ofthis protein, and in that its immunosuppressive and/or angiogenicproperties are inactivated to the extent of at least 70% by a physicaland/or chemical treatment, by genetic recombination or by an adjuvantmake-up, the said treatment preserving the property of being recognizedby antibodies directed against the said protein and preservingimmunogenic properties sufficient to generate antibodies whichneutralize or block the said native protein, with the exception ofcarboxymethylated Tat of HIV-1 or a fragment of this protein.

Of the above immunogenic compounds, the immunogenic compounds which arecharacterized in that they are derived from one of the followingimmunosuppressive and/or angiogenic proteins:

-   -   protein Tax of an HTLV-1 virus    -   protein E7 of the papilloma virus    -   mannan-dependent lectin produced by immunity cells, and in        particular activated T lymphocytes        are preferred

An immunogenic compound as defined above which is a product obtained bygenetic recombination and has a peptide homology of at least 70% withthe proteins Tat of HIV-1, Tax of HTLV-1 or -2 and E7 of HPV or themannan-dependent lectin produced by activated immunity cells, or with asegment of these proteins, is also preferred.

An immunogenic compound as defined above, characterized in that it istreated with an aldehyde, in that it is carboxamidated or in that it ismaleimidated is also preferred.

Finally, an immunogenic compound as defined above, characterized by anadjuvant make-up which renders it biologically inactive, such as an oilyemulsion in incomplete Freund's adjuvant (IFA) is preferred.

The desired immunogenic compound can also be derived from a homologousmutant.

The present invention also relates to a process for the preparation ofan immunogenic compound as defined above, characterized in that aninitially immunosuppressive or angiogenic protein which has a localaction and is induced by a cancer tumour or a homologous mutant proteinor a fragment of this protein is subjected to a physical or chemicaltreatment or to genetic recombination and the expected compound isselected and then purified, or a native or inactivated protein isincorporated into an adjuvant make-up. In fact, by galenical make-up ofa physiologically active protein, its biological activity can be masked,while preserving its immunogenicity.

The present invention in particular relates to a process for thepreparation of an immunogenic compound as defined above, characterizedin that a chemical treatment is carried out which comprises treatmentwith the aid of an aldehyde followed by coupling to a carrier protein,as well as a process, characterized in that a chemical treatment iscarried out which comprises a treatment which blocks the SH radicals bycarboxamidation, carboxymethylation or maleimidation.

The carboxymethylation reaction enables the thiol groupings (sulfhydrylgroupings) present in the cysteine radicals of the amino acid chains tobe modified. Carboxymethylation inactivates some toxic functionsdependent on SH groupings, as reported for the protein Tat by Frankel etal. Cell vol. 55 (1988). These groupings react with iodoacetic acid oriodoacetamide by an S-carboxymethylation or S-carboxamidomethylationreaction respectively.

The maleimidation reaction also blocks the SH groupings to formS-maleimide complexes.

For example, the protein Tat has 7 cysteines. These cysteinesparticipate in the formation of inter- and intra-chain disulphidebridges and contribute towards the formation of oligomers.

The reaction product is in each case an S-carboxymethylcysteinyl orS-carboxymethylamidocysteinyl radical.

A fragment can comprise 8 to 110 amino acids, for example, preferably 12to 60 amino acids, and in particular 12 to 40 amino acids. Such afragment can also comprise, from the C or N terminal sides, 1 to 5additional amino acids, that is to say different from the originalfragment. A fragment must also comprise at least one cysteine in orderto be able to undergo the carboxymethylation.

The carboxymethylation reaction can also be carried out with otherchemical agents, such as performic acid, 3-bromopropionic acid,ethyleneimine, (2-bromoethyl)-trimethylammonium bromide,2-bromoethanesulphonate, 1,3-propanesulphone etc.

Under the preferred conditions of carrying out the process describedabove, the said starting protein or fragment can be in a form which isfused to a marker (FP) or not fused (P). The FP form can modify per sethe molecular conformation and as a result modify its activity.

The starting proteins or fragment of the process are known products,inactivation processes of which may have been described by chance in theliterature, for example for a structural study, but which have in nocase been used as a vaccine, for example for the Tat of HIV-1 by FrankelA. D. et al. (Cellular uptake of the tat protein from humanimmunodeficiency virus, Cell, 1988, 55 : 1189–83), for Tat by Tanaka etal. Int. J. Cancer, 1991, vol 48, pages 623–630, or which have not yetbeen described, such as the inactivation of the protein E7 described bySpeidel et al, Eur. J. Immunol., 1997, 27, pages 2391–2399. Thesestarting proteins may even be commercially available (ImmunodiagnosticsInc., Cat# 1002-2) or may be prepared in a conventional manner.

In particular, the above starting proteins or fragments can be preparedby:

-   -   1) synthesis by genetic engineering or by biochemical synthesis;    -   2) purification.

By genetic engineering, the proteins produced can be purified byaffinity chromatography using, for example, antibodies directed againstthe protein or one of its fragments; it is also possible to synthesizethe protein fused to a marker (FP) for attachment to an affinity column.

Under other preferred conditions of carrying out the process describedabove, if the protein or fragment is fused to a marker (FP), it issubjected to:

-   -   concentration, for example by ultrafiltration;    -   desalination, for example by gel filtration;    -   a treatment with cyanogen bromide or enterokinase to cleave the        fusion protein and thus liberate the protein or fragment;    -   concentration and diafiltration;    -   cation exchange chromatography;    -   concentration by ultrafiltration, followed by exclusion gel        filtration.

The above reaction with cyanogen bromide allows thioethers to becleaved. The action of cyanogen bromide on the polypeptide molecules isselective, effecting cleavage at existing methionine radicals. Thisreaction leads to the formation of 2 polypeptide fragments permethionine radical. This reaction can be advantageously coupled with thecarboxymethylation reaction described above, but it is not necessary forthe inactivation.

Under other preferred conditions of carrying out the process describedabove, the expected protein or fragment is prepared in a form coupledwith a compound which allows its purification, for example a peptidefragment containing several histidines, preferably in a continuoussequence of 4, 5 or in particular 6 histidines or more, allowing fixingto a nickel column. To the extent that the presence of this compounddoes not induce toxicity and does not adversely modify theimmunogenicity of the protein or fragment, it is not necessary to cleaveit after purification. However, under preferred conditions of carryingout the invention, this compound is cleaved to eliminate it.

The modified proteins which are initially in particularimmunosuppressive and/or angiogenic proteins which have a local actionand are induced by cancer tumours, and fragments, and of which theimmunosuppressive properties are inactivated by a suitable treatment, towhich the present invention relates, have very interestingpharmacological properties. In particular, they have remarkableproperties antagonistic towards the properties of immunosuppressiveand/or angiogenic proteins which have a local action and are induced bya cancer tumour.

These properties are illustrated below in the experimental part. Theyjustify the use of the modified proteins described above as amedicament.

The invention thus also relates to medicaments, characterized in thatthey are made up of immunogenic compounds as defined above, for theiruse in a method of therapeutic treatment of the human or animal body, aswell as the use of such an immunogenic compound for the preparation of acurative or preventive medicament intended for neutralization of theimmunosuppressive and/or angiogenic effects of the aboveimmunosuppressive and/or angiogenic proteins, and in particular of theprotein Tat of HIV-1, the protein E7 of the papilloma virus and theprotein Tax of HTLV-1. In fact, the compounds according to the inventionhave lost their immunosuppressive properties or their angiogenicproperties and can thus be administered to humans, as will be seen belowin the experimental part.

The medicaments according to the present invention are employed, forexample, in curative treatment of cancers, in particular of cancersinduced by viruses such as, for example, the ATL (acute T cellleukaemia) caused by HTLV-1, or the cancer of the cervix caused by thepapilloma virus, or the Burkitt's lymphoma or Kaposi's sarcoma caused byviruses of the herpes family, Epstein-Barr (EBV) and HHV-8 respectively.

The immunogenic compounds according to the invention can be used asfollows:

An immunogenic compound according to the present invention isadministered to a patient, for example by the subcutaneous orintramuscular route, in an amount sufficient to be effective at thetherapeutic level, to a subject having need of such a treatment. Thedose administered can range, for example, from 10 to 1,000 μgsubcutaneously, once a month for three months, and then periodically asa function of the serum level of the antibodies induced, for exampleevery 2–6 months. Two or more different immunogenic molecules could beadministered in the same preparation to induce antibodies whichneutralize all the deleterious functional sites in the case where asingle immunogenic molecule does not carry all the active sites of thetoxin or the over-produced cytokine which is to be neutralized.

The invention also relates to pharmaceutical compositions comprising atleast one abovementioned immunogenic compound as the active principle.

As medicaments, the immunogenic compounds of the invention can beincorporated into pharmaceutical compositions intended for anyconventional route in use in the field of vaccines, in particular thesubcutaneous route, the intramuscular route, the intravenous route orthe oral route. The administration can take place in a single dose or adose repeated once or several times after a certain interval of time.

The present Application thus also relates to a curative or preventivepharmaceutical composition, characterized in that it comprises, as theactive principle, one or more new immunogenic compounds as definedabove, that is to say with the exception of the inactivated Tat proteinof HIV-1, or its fragments. The immunogenic compound can be made up byitself or as a mixture with an excipient or mixture of pharmaceuticallyacceptable excipients, such as an adjuvant.

The present Application more particularly relates to a vaccinecomprising, as the immunogen, an immunogenic compound defined above, andin particular an initially immunosuppressive and/or angiogenic proteinwhich has a local action and is induced by a cancer tumour, or afragment of this protein, the immunosuppressive and/or angiogenicproperties of which are inactivated to the extent of at least 70% by asuitable treatment.

The present invention also relates to a process for the preparation of acompound described above, characterized in that the active principle orprinciples is or are mixed with the acceptable, in particularpharmaceutically acceptable, excipients by methods which are known perse.

Administration of immunogenic compounds according to the invention to apatient corresponds to an active immunotherapy. It may also be ofinterest to proceed with a passive immunotherapy, that is to say toprovide a patient directly with the antibodies he requires to neutralizethe harmful effects of the above proteins, for example immunosuppressiveproteins which have a local action and are induced by tumours.

These antibodies, for example against immunosuppressive and/orangiogenic proteins, can be obtained conventionally and, by way ofexample, after immunization of a mammal, human or animal, with the aidof an immunogenic compound as defined above, by cloning human Blymphocytes transformed by the Epstein-Barr virus and then collectingthe expected antibodies secreted by the said transformed B lymphocytes,or by genetic recombination from a phage library.

The present Application thus also relates to such processes for thepreparation of antibodies against proteins, in particularimmunosuppressive and/or angiogenic proteins of a cancer tumour, and inparticular antibodies against protein E7 of the papilloma virus oragainst protein Tax of HTLV-1, and in particular a process for thepreparation of an above antibody, characterized in that a mammal isimmunized with the aid of an immunogenic compound as defined above andthe antibodies formed are then collected.

The present Application also relates to an antibody against animmunosuppressive or angiogenic protein of a cancer tumour, and inparticular polyclonal or monoclonal antibodies obtained from mammalsimmunized with an immunogenic compound defined above, and especially animmunosuppressive or angiogenic protein of a cancer tumour which hasbeen biologically inactivated but is immunogenic, in particular theprotein E7 of the papilloma virus or the protein Tax of HTLV-1, or theirfragments. These passively administered antibodies, which are eitherallogenic (human) or xenogenic (animal), could be complete monoclonal orpolyclonal antibodies or F(ab′)2 or Fab fragments of the antibody.

“Antibodies against an immunosuppressive or angiogenic protein of acancer tumour” are understood as meaning monoclonal or polyclonalantibodies or F(ab′)2 or Fab fragments of these antibodies or antibodiesagainst an immunosuppressive or angiogenic protein of a cancer tumourwhich are obtained by genetic construction from a phage library.

The xenogenic antibodies originate from animals hyperimmunized with animmunogenic compound according to the invention, in particular with theprotein E7 of the papilloma virus or the protein Tax of HTLV-1 or itsderivatives (peptide fragments of the protein E7 of the papilloma virusor of the protein Tax of HTLV-1 detoxified according to the invention),and are

-   -   either polyclonal, originating from hyperimmunized animals,    -   or monoclonal, obtained after hybridization by the Kohler and        Milstein technique from splenic cells or adenocytes with a        myelomatous line, type x63, in particular x63AG3. In this case,        horse or rabbit antibodies are preferred.

The present Application also relates to a process for the preparation ofantibodies against an immunosuppressive or angiogenic protein of acancer tumour, characterized in that a mammal, human or animal, isimmunized with an immunogenic compound as defined above.

The present invention also relates to a process for the preparation ofmonoclonal antibodies against an immunosuppressive or angiogenic proteinof a cancer tumour, characterized in that B cells originating fromindividuals immunized with an immunogenic compound according to thepresent invention are used, the said B cells being transformed by theEBV virus and producing specific antibodies against an immunosuppressiveor angiogenic protein of a cancer tumour.

The above EBV+ cells can be cultured to produce the expected antibodies.These cells, as has been seen, originate in particular from patientsimmunized with a native immunosuppressive or angiogenic protein of acancer tumour, or with an immunogenic compound according to theinvention.

The present Application also relates to a process for the preparation ofmonoclonal antibodies according to the invention directed against animmunosuppressive or angiogenic protein of a cancer tumour, which is oris not effectively immunosuppressive, characterized in that hybridomasof a mammal, in particular of mice, from splenocytes or adenocytes, inparticular of mice immunized with a native immunosuppressive orangiogenic protein or an immunogenic compound according to theinvention, and myeloma cells, preferably of the line x63, are preparedby processes well-known in the prior art (Kohler and Milstein).

The present Application also relates to a process for obtainingantibodies against an immunosuppressive or angiogenic protein of acancer tumour by the technology of genetic recombination, characterizedin that an immunogenic compound as defined above is used as theimmunogen.

The present Application also relates to F(ab′)2 or Fab fragments of thesaid antibodies; these can be obtained by enzymatic digestion, forexample.

The present invention similarly relates to a process for passiveimmunization of cancer subjects using specific antibodies against animmunosuppressive or angiogenic protein of a cancer tumour, andspecifically against the protein E7 of the papilloma virus or againstthe protein Tax of HTLV-1, neutralizing or blocking the harmful effectsof this protein and being capable of preparation as indicated above, orF(ab′)2 or F(ab) fragments of these antibodies.

The present Application also relates to a process for activeimmunization, characterized in that an immunogenic compound as definedabove, advantageously combined. with a mineral, oily or syntheticimmunity adjuvant, or an immunogenic compound as defined above,advantageously coupled, for example with the aid of a dialdehyde, orcombined with a protein which increases its immunogenicity, is used asthe immunogen.

These immunizations can be carried out both curatively and preventively.

A derivative of the protein E7 of the papilloma virus or of the proteinTax of HTLV-1 is preferably used as the immunogens for all the processesabove and below.

The invention also relates to a pharmaceutical composition comprising,as the curative or preventive active principle, at least one antibodyagainst an immunosuppressive or angiogenic protein of a cancer tumour asdefined above or obtained by the above processes.

Finally, the invention relates to the use of an above immunogeniccompound or antibody for the preparation of a medicament intended fortreatment of the local immunosuppressive effects of a protein, which hasa local immunosuppressive or an angiogenic action, of a cancer tumour.

Summarizing, and in particular, the present invention relates to thepreventive or curative use in a cancer subject of specific antibodiessuch as to block the action, in particular immunosuppressive, of aprotein, which has a local immunosuppressive or an angiogenic action, ofa cancer tumour, and in particular of the protein Tat of HIV-1, of theprotein E7 of the papilloma virus or of the protein Tax of HTLV-1. Thesespecific antibodies could originate:

-   -   1. from the subject himself, induced by an active immunization        (vaccination) with a local immunosuppressive protein of a cancer        tumour deprived of the said immunosuppressive effects but        immunogenic (the properties capable of inducing the formation of        antibodies have been preserved, while the protein is present and        prepared in a suitable manner, coupled or not coupled to a        “carrier”, aggregated or not aggregated, in the presence or        absence of an adjuvant) or    -   2. from a foreign allo- or xenogenic organism, administered to        the subject by passive immunization (serotherapy). These        antibodies administered passively could be complete monoclonal        or polyclonal antibodies or F(ab′)2 or Fab fragments of the        antibody.

The invention also relates to pharmaceutical compositions.

-   -   a) A pharmaceutical composition comprising, as the preventive or        curative agent, an initially local immunosuppressive or        angiogenic protein of a cancer tumour but deprived of the said        immunosuppressive and immunogenic effects, according to the        invention.    -   b) A pharmaceutical composition comprising, as the preventive or        curative agent, antibodies against a protein, which has a local        immunosuppressive or angiogenic action, of a cancer tumour,        which antibodies are produced from organisms immunized against        the said protein, or its F(ab′)2 or Fab fragments, according to        the invention.

The invention also provides a kit comprising a vaccinal pharmaceuticalcomposition which, in addition to the active principle (initially localimmunosuppressive or angiogenic protein of a cancer tumour, but deprivedof the said immunosuppressive or angiogenic effects and immunogenic, orits derivative or antibodies against a local immunosuppressive orangiogenic protein of a cancer tumour), can comprise an adjuvant and/oranother immunogen having anticancer properties.

Finally, the invention provides a pharmaceutical composition in aconventional galenical form. In particular, the active principleaccording to the invention is combined, in an amount sufficient to beeffective from a therapeutic point of view, with a diluent or a carrierwhich is acceptable from the pharmaceutical point of view.

To reduce the immunosuppressive load produced by the tumour and toimprove still further the immune response, this anti-suppressiveimmunization could be combined with more conventional means aimed atreducing the size of the tumour, such as chemotherapy, radiotherapy,surgical excision or addition of tumour suppressor genes provided by thetechniques of gene therapy (DNA carried by viral vectors, lipid vectorsetc.) or such as active immunization against proteins without a localimmunosuppressive or angiogenic action, such as MAGE or proteins of astructure such as those of the papilloma virus.

It could also be possible to combine other anti-suppressive and/or-angiogenic immunizations, for example by immunizing against cytokinesor lectins capable of mediating the suppressive action on the immunitycells, or to combine immunizations against conventional tumoral antigens(non-immunosuppressive or -angiogenic) capable of increasing the cellimmunity, in particular killer, response (CTL cells or NK cells)directed against the tumour cells or cells infected by viruses. Theadvantage of these combinations is that they would allow the immunesystem to respond better to anti-immunosuppressive immunization andconsequently to restore itself to a better degree.

Summarizing, immunization against a paracrine immunosuppressive orangiogenic factor in an inactive but still immunogenic form could becombined with more conventional methods, such as radiotherapy,chemotherapy, surgical excision or treatment with suppressor genes orimmunizations against cytokines or lectins produced by immunity cells (Tcells or APC) which mediate the immunosuppressive and/or angiogenicaction or against tumoral antigens.

In fact, in some cancers of the same viral origin, soluble factors ofcell origin, such as cytokines or lectins, can also play a role locallyas a mediator of immunosuppression and/or angiogenesis within thetumour. This is the case with IFNα, an immunosuppressive cytokin,over-produced locally within lymphoid tissues infected by HIV in casesof the AIDS disease.

In vitro experiments on blood mononuclear cells (PBMC) infected withHIV-1 has shown that the protein Tat was involved in the over-productionby the APC of the immunosuppressive cytokine IFNα. Interestingly, theprotein E7 of HPV, like the protein Tat of HIV-1, inducesover-production of immunosuppressive IFNα by the APC.

Consequently, the escape from the immune system and/or the angiogenesisof cancers could also be combated by inducing or administeringantibodies directed specifically against the cytokines, over-productionof which is responsible for immunopathogenesis, in particularimmunosuppression, such as IFNα, and/or angiogenesis, such as TFNα, inaccordance with the international patent application WO 92/225.

The immunosuppressive effects due to over-production of IFNα in cancersof the AIDS disease and of the cervix, like those due to over-productionof TGFB in virus-induced gliomas, could thus be blocked by antibodiesdirected against these natural cytokines induced by an activeimmunization (vaccination) using cytokinoids (modified cytokines whichare biologically inactive but immunogenic) as the vaccine. Suchantibodies could also be administered passively (passive immunization).

FIG. 1 shows the anti-Tat responses of 5 subjects immunized inexperiment 5 below, determined by ELISA on plates referenced by thenative protein Tat, after immunization with the Tax toxoid in the 5subjects and the various repeats.

FIG. 2 shows the antibody titres obtained after all the immunizations,in comparison with 2 non-immunized controls.

FIG. 3 shows the induction of inducible NO synthase (iNOS) expression inhuman vascular endothelial cells (HUVEC) pretreated by the proteinHPV₁₆E7 and cultured in the presence of cytokines TNFα, IL-1 and IFNγ.

FIG. 4 shows the induction of the messenger RNA of iNOS after 6 hours oftreatment with the protein E7 (100 ng/ml) and the cytokines: TNFα (5ng/ml), IL-1β (1 ng/ml) and IFNγ (1,000 U/ml).

FIG. 5 shows the results of the flow cytometry of human endothelialcells (HUVEC) pretreated or not pretreated with the oncogenic protein E7and cultured for 12 hours in the presence of cytokines IFNγ, TNFα andIL-1 (effect of the protein E7 on expression of the 1 ICAM-1 adhesionmolecule in the endothelial cells of the human umbilical cord).

The examples and experiments which follow illustrate the presentapplication.

EXAMPLE 1 Production of the Protein Tat Inactivated by ChemicalTreatment

Preparation

2 ml of bacteria deposited at the Collection Nationale de Cultures deMicro-organismes [National Collection of Microorganism Cultures] inParis on 26 Dec. 1997 under no. 1 1964, which is an E. coli bacteriuminto which has been inserted by transfection a recombinant plasmidcontaining a gene which codes for a polypeptide containing a fragmentmade up of 6 histidines, combined with the gene of the protein Tat, arecultured in 400 ml of a base culture medium (yeast extract 5 g/l,tryptone 1 g/l, sodium chloride 1 g/l) and 40 ml of a solution 1(CaCl₂2H₂O: 0.175 g/l, MgSO₄7H₂O: 5.9 g/l, glucose: 6 g/l). The cultureis then incubated at 30° C. for 10 hours at 250 rpm.

The above prefermentation is followed by a fermentation under analogousconditions, maintaining the level of dissolved oxygen at a level of 70%of saturation by regulating the stirring. When the optical densitymeasured at 650 nm reaches the value 6, 100 ml of a sterile 3.75%solution of IPTG (isopropyl β-D-thiogalactopyranoside) in deionizedwater are added. At the same time, the medium is supplemented with asterile 25% solution of yeast extract. A solution comprising MgSO₄7H₂O:8.5 g/l, glucose: 300 g/l, (NH₄)₂SO₄: 106 g/l, trace elements, is thenadded, maintaining the glucose concentration above 2 g/l. The bacterialmass is then harvested 4 hours after introduction of the IPTG.Diafiltration is carried out against a buffer of tris 0.1 M, NaH₂PO₄ 0.1M, dithiothreitol 1 mM, pH 8.0, by counter-current filtration (threshold0.3 μm).

Three litres of the above crude product are lysed by 4 passes under anincreased pressure of 500 bar. The lysate is then centrifuged for 15 minat 4° C. and 5,000 rpm. An amount of urea sufficient to obtain an 8 Msolution is then added to the supernatant and the pH is adjusted to 8.

Purification is then carried out by affinity chromatography over anickel-NTA agarose column (Qiagen), equilibrated beforehand with 600 mlof buffer A (urea 8M, NaH₂PO₄ 0.1 M, tris-HCl 0.1 M, DTT 1 mM, pH 8.0).When charged, the column was washed with 250 ml of buffer A. The proteinwas then eluted using a discontinuous gradient to obtain the expectedfusion protein.

The solution obtained in this way is concentrated on an Amicon membraneat a cut-off threshold of 3,000 daltons to give a final concentration of10 mg/ml. A crude eluate comprising the expected recombinant fusionprotein of HIV-1 Tat is obtained in this way.

A Tat fusion protein fused genetically with a peptide fragment rich inhistidines, which allows its attachment to nickel for the purpose ofpurification, was produced in this way.

The eluate obtained in this way will be subjected to thecarboxymethylation reaction.

Preparation of the Tat Toxoid

Stage A: Carboxymethylation

A solution of the protein Tat fused to a peptide fragment rich inhistidine, prepared in preparation 1 above, is adjusted to obtain thefollowing concentrations: urea 8 M, Tris HCl 0.3 M, dithiothreitol 10mM, pH 8.4.

2.6 g iodoacetic acid are added to 100 ml of the above solution under aninert atmosphere. This solution is incubated at 37° C. with exclusion oflight and under an inert atmosphere for 90 min. The reaction is blockedby addition of 1 ml of 98% β-mercaptoethanol and the incubation iscontinued for 60 minutes under the same conditions as above.

The solution resulting from the preceding stage is concentrated with theaid of an Amicon concentrator (Cat# 8400) over a YM3 membrane (thresholdof 3,000 D) to a concentration of 10 mg/ml.

The product is then desalinated by passage through a Cellufine GH25column (MATREX), equilibrated with the aid of 300 ml of a solution ofurea 4 M, 0.1 M HCl.

Stage B—Purifications

The eluate is then subjected to ultrafiltration and treated withcyanogen bromide to cleave the amino-terminal part of thecarboxymethylated product at the level of the methionine. Cyanogenbromide is added in excess under an inert atmosphere in a proportion ofabout 50 moles of cyanogen bromide per mole of methionine. This solutionis kept in a closed container for 24 h at 37° C. The excess cyanogenbromide is driven off by evaporation under reduced pressure.

The solution obtained is then concentrated, subjected to diafiltrationagainst acetic acid-sodium acetate buffer, 0.05 M, pH 5 using an Amicondiafilterer fitted with a YM3 membrane (threshold 3,000 KD). The amountof inactive product obtained in solution is of the order of 450 mg.

This solution is filtered over an SP-Sepharose FF ion exchange column,equilibrated beforehand with 200 ml of buffer A, and the product iseluted by an NaCl gradient.

The eluate is concentrated again by diafiltration in the same Amiconsystem and the fraction obtained is purified by gel filtration over aSephacryl S column (Pharmacia), equilibrated with phosphate buffer, pH7.4. The end product is filtered over a membrane at 0.22 μm and storedat 4° until used.

This product was subjected to the analyses below and was the subject ofthe pharmacological tests.

Analyses

-   -   Total amount of inactivated product analysed by the Bradford        test: 150 mg.    -   Electrophoresis over polyacrylamide gel,        sulphododecylsulphate-PAGE, coloration silver (Phast System,        Pharmacia, homogeneous 20% gel): single band with a molecular        weight compatible with that expected. No other bands detectable        at higher or lower weights.    -   Electrophoresis by isoelectric gradient (Phast System,        Pharmacia, GEL IEF 3-9): a single band visible.    -   Western blot (Phast System, Pharmacia, monoclonal antibody of        hybridoma 5G11): a single band observed and absence of        aggregated or degraded material.    -   Biological activity (induction of the CAT gene in the line        HELA-HIV-1-LTR CAT): no activity detectable.    -   Sequencing of the N-terminal part using the standard sequencing        technique of Edman with an Applied Biosystem sequencer (model        477A) the first 20 amino acids obtained coincide with the        expected sequence.        E-P-V-D-P-R-L-E-P-W-K-H-P-G-S-Q-P-K-T-A  (SEQ ID NO:1)    -   Endotoxins (LAL test in accordance with the protocol of USP        XXIII): less than 0.5 endotoxic units per mg of protein.    -   Sterility (in accordance with the protocol of USP XXIII):        corresponds to standards.    -   Measurement of the degree of substitution: using Ellman's        reagent to determine the free sulfhydryl groupings by comparison        with a standard curve measuring cysteines. Comparing the native        Tat with the carboxymethylated Tat, it is found that only 0.03%        of the cysteines remained active in the carboxymethylated Tat,        that is to say 1 active cysteine per 3,330 inactive cysteines. A        simple calculation shows that to have a Tat molecule with 7        active cysteines under these conditions, several kg of the        protein Tat would be necessary. The inactivation is thus        virtually total.

This method leads to the production of non-immunosuppressive Tat, calledTat toxoid, as will be seen below.

EXAMPLE 2 Preparation of Mutant Tat Obtained by Genetic Recombination,which is Inactive but Immunogenic

The mutant (AA25; Cys versus Gly) is obtained either by mutagenesisusing the phage M13 or by a PCR. This mutant, in contrast to thewild-type Tat protein, proved to be inactive in an amount of 1 μg per mlin the CAT assay and in the immunosuppression test on peripheral bloodmononuclear cells stimulated by memory antigens (PPD or tetanus toxoid).

EXAMPLE 3 Preparation of the Protein Tat in an Oily Make-Up Rendering itBiologically Inactive.

4 ml of oil (incomplete Freund's adjuvant or ISA 51) are pipetted into aflask cooled in an ice-bath. The shaft of a homogenizer, Silverstontype, is introduced into the oil, avoiding contact with the walls of theflask. 4 ml of immunogen in aqueous solution, in a fraction of 20 μl forthe 1st ml, 250 μl for the 3rd ml and 500 μl for the 4th ml, are thenadded to the oil. Between each addition the Silverston is activated at8,000 revolutions per minute for 4 to 5 seconds, and after addition ofall of the aqueous solution for 6 minutes at 8,000 revolutions perminute. A stable water-in-oil emulsion is obtained in this way. In thismake-up, the protein Tat, although still immunogenic, proves to bebiologically inactive. In vivo, subcutaneous administration of anemulsion comprising 100 μg of Tat has harmful effects neithersystemically (curve of weight:temperature) nor on the immune system(normal proliferative cell response of splenocytes stimulated by thememory antigens). In vitro, the protein Tat contained in the aqueousphase isolated from the emulsion proved to be antigenic by the ELISAtest but functionally inactive in the CAT assay test.

EXAMPLE 4 Production of the Protein E7 of HPV (Strain 16) by Cells ofthe Line SIHA, the Genome of which Contains One Copy per Cell of the DNAwhich Codes for this Protein.

Cells of the line SIHA cultured in MEM medium comprising 10% FCS areconcentrated to 5×10⁶/ml and incubated with or without serum at 37° C.in the presence of 5% CO₂ for 18 hours. At the end of this incubationthe viability of the cells is greater than 85%. On a smear, after Giemsastaining, these cells are morphologically normal. With reference tocells before incubation, their cytoplasm looks more basophile, thenucleus accommodates one or more large nucleoli and the mitoses are lessnumerous or non-existent. These cells export into the culturesupernatant measurable amounts of the protein E7 which are identifiedspecifically by the immunoblot test using rabbit antibodies against E7revealed by ELISA. With reference to immuno-prints made on knowndecreasing amounts of purified recombinant protein E7, these cells inculture have produced amounts of E7 greater than 10 pg/ml in theextracellular medium.

EXAMPLE 5 Production of Protein E7 Inactivated by Chemical Treatment

Tests using glutaraldehyde have allowed production of a protein E7 whichis inactivated in the immunosuppression tests. Treatment withglutaraldehyde is carried out in the following manner: to a solution ofE7 of 1 mg/ml in disodium phosphate, 70 mM at pH 8.2, is addedglutaraldehyde at a final concentration of 0.026 M or 0.0026 M. Thereaction is allowed to proceed over varying periods of time ranging from1 min to 3 hours at room temperature. At the end of each reaction timethe reaction is blocked by addition of glycine at a final concentrationof 100 mM. The various samples are dialysed for 16 h at 4° C. against100 times their volume of PBS. The dialysis is repeated twice. It isfound in all cases that from after 1 hour of treatment withglutaraldehyde the immunosuppressive of E7 has disappeared, as measuredin the immunosuppression test. It is appropriate to optimize thereaction conditions in order to keep the immunogenicity of the proteinat a maximum: this is effected by testing the preparations by the methodused in experiment 3.

Tests were also carried out with formaldehyde at a final concentrationof 33 mM and the inactivation is tested at from 1 hour to several days.The reaction is blocked by addition of glycine, before repeated dialysisagainst PBS is carried out. The protein obtained, or the E7 toxoid, incontrast to the native protein, showed no immunosuppressive activity inthe activity test on peripheral blood mononuclear cells activated bymemory antigens (PPD and tetanus toxoid).

EXAMPLE 6 Production of Protein Tax Inactivated by Chemical Treatment

The protein Tax was produced by genetic engineering and obtained at aconcentration of 1 mg/ml. Use of the same inactivation protocol usingcarboxamidation led to the production of a product derived from the Taxtoxoid which is inactive in the immunosuppression test on bloodmononuclear cells stimulated by memory antigens (PPD and tetanustoxoid).

EXAMPLE 7

An injectable composition was prepared, corresponding to the  50 μgformula composed in example 1 excipient q.s. for a final vial of 0.5 ml(detail of the excipient: ISA 51 adjuvant (Seppic))

EXAMPLE 8

An injectable composition was prepared, corresponding to the 100 μgformula composed in example 3 excipient q.s. for a final vial of 1 ml  1ml (detail of the exigent: aluminium hydroxide (Superfos, Kvistgaard,DK))Pharmacological StudyExperiment 1a. Immunosuppressive Properties of Extracellular ProteinFactors Originating from Cancer Cells, Cells Infected by Viruses orImmunity Cells.

1a1. The protein Tat of HIV-1 was purified at a concentration of 1 mg/mlas prepared by Frankel A. D. et al. (Cellular uptake of the Tat proteinfrom human immunodeficiency virus, Cell, 1988, 55: 1189–93).

Peripheral blood mononuclear cells (PBMCs) are taken from a healthysubject, purified by the Ficoll method and cultured at 1 million cellsper ml in microwells in the presence of PPD memory antigens (Mérieux,dilution to 1/1,000) in RPMI, BSA serum 10%. The protein Tat is added ina decreasing dose: 10 mg/ml, 3 mg/ml, 1 mg/ml etc. To obtain morepronounced results, it is sometimes necessary to preincubate the cellsfor 2 h in RPMI with the Tat protein before addition of the serum, sincethe protein can sometimes bond to components of the serum and thusinactivate its capacity to act on the cells.

IL-2 is added after 48 h. Finally, after 5 days the cell proliferationis measured in the wells by the test of incorporation of tritiatedthymidine. The results are the following:

Proliferation of the PCBMs (CPM) Without addition of Tat 28,000 Tat (10μg/ml)  8,000 Tat (3 μg/ml) 13,000 Tat (1 μg/ml) 21,000 Tat (0.3 μg/ml)26,0001a2. Immunosuppressive cytokines covered by the international patent WO92/225.

1a3. Immunosuppressive lectins. We have recently identified a lectinproduced by T lymphocytes, the hapten of which is mannan and which isimmunosuppressive. The inhibition of the cell immune response isdemonstrated 1) by the increase in the production of IFNα, animmunosuppressive cytokine, by the APC. This increase is blocked in thepresence of mannan and not blocked in the presence of lactose orfructose; 2) by the inhibition of the proliferation of T lymphocytes,PBMC stimulated by memory antigens (PPD, tetanus toxoid) or byalloantigens.

Experimentally, this lectin was demonstrated in the supernatants of H9cells cultured on HL1 medium without serum, or of PBMC preactivated bythe cytokine IL-2 and cultured on HL1 medium without serum. The culturesupernatants were concentrated by diafiltration over a membrane with acut-off at 10 kDa. These concentrated supernatants have the followingproperties:

-   -   1) agglutination of H9 cells inactivated by mytomycin; this        agglutination is prevented by mannan.    -   2) stimulation, at a low concentration, of the proliferation of        H9 cells.    -   3) inhibition, at a high concentration, of the proliferation of        H9 cells.

Agglutination of fixed cells by supernatants of H9 or PBMCs Dilutions ofthe supernatants 1/2 1/4 1/8 1/16 1/32 1/64 1/28 HL1 medium > 30 kDa  0*0 0 0 0 0 0 HL1 medium < 30 kDa 0 0 0 0 0 0 0 H9 SN > 30 kDa 4 4 3 2 2 10 H9 SN < 30 kDa 0 0 0 0 0 0 0 PBMC SN > 30 kDa 4 3 2 1 0 0 0 PBMC SN <30 kDa 0 0 0 0 0 0 0 *Agglutination scores

Proliferation of H9 cells in the presence of supernatants rich in lectinDilutions of the supernatants 0 1/2 1/4 1/8 1/16 HP SN > 30 kDa  12,000* 8,000 16,000 21,000 13,500 HP SN < 30 kDa 12,000 12,500 11,800 12,00012,500 *Measured by the test of incorporation of 3H-thymidine (cpm).

These properties allow the factor present in the supernatants due to alectin to be identified.

The immunosuppressive properties of this lectin could have been relatedto the fact the action of these supernatants on the APC (dendriticcells, macrophages) in culture activated by IFNγ is objectified by ahigh increase in the production of IFNα by these cells. However, inparallel with the increase in the production of IFNα, the loss ofantiviral potency of IFNα on the VSV when the MDBK cells are pretreatedby the culture supernatants is observed.

Culture supernatant Neutralization on MDBK (%) control medium 100 H9 50H9 mannan 100 PBMC 40 PBMC mannan 80

Increase in the production of Culture supernatant IFNα by activated APC(%) control medium 100 H9 250 PBMC 3001a4. Case of the Protein E7 of the Papilloma Virus.

The same experiment as with Tat is carried out with the protein E7 ofthe papilloma, virus, purified by the method of Speidel et al, Eur. J.Immunol, 1997, 27, pages 2391–2399.

The tests of incorporation of 3H-thymidine gave similar results.

Proliferation of PBMCs (CPM) Without addition of E7 29,000 E7 (10 μg/ml) 6,000 E7 (3 μg/ml) 10,000 E7 (1 μg/ml) 11,000 E7 (0.3 μg/ml) 16,0001a5. Case of the Protein Tax of HTLV-1.

The same experiment as with Tat was also carried out with the purifiedprotein Tax of HTLV-1, obtained by the method of Tanaka et al. Int. J.Cancer, 1991, vol 48, pages 623–630.

The tests of incorporation of 3H-thymidine gave similar results.

Proliferation of PBMCs (CPM) Without addition of Tax 25,000 Tax (10μg/ml)  7,000 Tax (3 μg/ml) 15,000 Tax (1 μg/ml) 21,000 Tax (0.3 μg/ml)26,000

These results show that the above native proteins are capable ofinducing a dose-dependent immunosuppression and have the capacity fordemobilizing the immune system. Angiogenic properties of the protein E7.

Experiment 2. Angiogenic Properties of Extracellular Protein Factors.

The purified protein E7 of HPV (strain 16), which significantlyincreases the production of the cytokines TNFα and IL-1 by macrophagesand dendritic cells, also showed angiogenic properties within culturesof endothelial cells originating from the human umbilical cord (HUVEC).In fact, we found that the action of the protein E7 on these cellscultured in a medium comprising EGF, stimulated or not stimulated by thecytokines TNFα, IFNγ or IL-1β, manifested itself by a significantincrease in the level of production of NO synthases, as was establishedby FACS in fluorescence cytometry (FIG. 1) or by a PCR after extractionof the total RNA of cells treated with the protein E7 or the cytokines(FIG. 2). It was also shown that the protein E7, either by itself or incombination with cytokines, significantly increased expression by thesesame cells of ICAM-1 adhesion molecules (FIG. 3).

Experiment 3. Absence of Suppressive Activity of Toxoids in theImmunosuppression Test.

The proteins modified by chemical treatment of examples 1, 2 and 3 weretested under the same conditions as those described in experiment 1.

No immunosuppressive effect could be demonstrated for these products:

Proliferation of the PBMCs (% with respect to the control) Control 100Inactivated Tat (10 μg/ml) 95 Native Tat (10 μg/ml) 20 Inactivated E7105 Native E7 25 Inactivated Tax 100 Native Tax 25Experiment 4. Immunogenicity of the Proteins of Examples 1, 2 and 3

Toxoids prepared according to examples 1, 2 and 3 were used to immunizemice.

The immunization protocol is that conventionally used: The mice areinjected (im) with 100 μl of an emulsion (1:1) in complete Freund'sadjuvant comprising 20 μg of the product on day 0 with a repeat inincomplete Freund's adjuvant of 5 μg on days 21 and 35. The serum of themice is sampled on days-2 and 40 and analysed by ELISA on platessensitized with the corresponding native protein (not chemicallytreated) and with the toxoids prepared in examples 1, 2 and 3. The serawere tested at a dilution to 1/500.

The results obtained were comparable for the 3 products tested here, Tattoxoid, Tax toxoid and E7 toxoid.

These results, expressed in optical density, obtained on 3 immunizedmice (1 to 3) and 3 non-immunized mice (4 to 6) are shown in thefollowing table:

Native protein Inactivated protein Mouse 1 D-2 0.2 0.2 D40 2.1 2.1 Mouse2 D-2 0.15 0.2 D40 2.2 2.2 Mouse 3 D-2 0.2 0.1 D40 1.9 2.1 Mouse 4 D-20.15 0.15 D40 0.1 0.1 Mouse 5 D-2 0.1 0.1 D50 0.1 0.15 Mouse 6 D-2 0.20.2 D40 0.1 0.1

These results show that the mice immunized by the toxoid produceantibodies capable of recognizing the native protein and the toxoid inthe same manner. On the other hand, this confirms the innocuity of theimmunization with the toxoids, since the mice tolerated the immunizationvery well.

Experiment 5. Immunization of Humans with the Product of Example 1.

5 seronegative subjects were subjected to immunization with theinactivated immunogenic protein Tat of example 1 in incomplete Freund'sadjuvant (ISA 51, Seppic) followed by a first repeat about 3 weeks laterand a second repeat about 15 days later. An immune response against thenative protein Tat could be objectified by ELISA tests. FIG. 1 shows theanti-Tat responses of the 5 subjects immunized, determined by ELISA onplates referenced with the native protein Tat, after immunization withthe Tat toxoid on the 5 subjects and the various repeats. FIG. 2 showsthe titres obtained after all the immunizations, compared with 2non-immunized controls.

It can thus be seen that the toxoid can be used on humans and causes animmune response which produces a cross-reaction with the native proteinTat.

1. A method for inhibiting the proliferation of cancer cells in apatient, comprising: inactivating a polypeptide that is a soluble factorwhich is released or induced by cancer cells or by cells infected by acancer-inducing virus and has a local immunosuppressive and/orangiogenic activity with respect to the cancer cells to be inhibited, byphysical and/or chemical treatment, and/or by modification of the aminoacid sequence thereof, so as to eliminate at least 70% of theimmunosuppressive and/or angiogenic activity of the non-inactivatedfactor and yet to retain sufficient immunogenic properties to raiseantibodies that neutralize the immunosuppressive and/or angiogenicactivity of the factor; and administering said inactivated protein tothe patient in a manner such that antibodies thereto are produced in aquantity sufficient to combine with the factor and to neutralize theimmunosuppressive and/or angiogenic activity of the factor, therebyinhibiting the proliferation of the cells, wherein said factor isprotein E7 of the papillomavirus.
 2. A method in accordance with claim1, wherein said inactivating step includes a physical treatment selectedfrom the group consisting of heat treatment, UV irradiation, X-rayirradiation, and contact with an atmosphere rich in O₂.
 3. A method inaccordance with claim 1, wherein said inactivating step includes achemical treatment selected from the group consisting of treatmentcarried out with the aid of a coupling agent, treatment carried out withthe aid of a carrier protein activated by pretreatment with adialdehyde, treatment with a monoaldehyde, carboxymethylation,carboxyamidation, maleimidation, and galenical presentation with an oilyliquid.
 4. A method in accordance with claim 3, wherein the chemicaltreatment is treatment carried out with the aid of a coupling agent. 5.A method in accordance with claim 4, wherein said coupling agent is adialdehyde.
 6. A method in accordance with claim 5, wherein saiddialdehyde is glutaraldehyde.
 7. A method in accordance with claim 3,wherein the chemical treatment is treatment with a monoaldehyde.
 8. Amethod in accordance with claim 7, wherein said monoaldehyde isformaldehyde.
 9. A method in accordance with claim 3, wherein thechemical treatment is carboxyamidation.
 10. A method in accordance withclaim 3, wherein the chemical treatment is carboxymethylation.
 11. Amethod in accordance with claim 3, wherein the chemical treatment ismaleimidation.
 12. A method in accordance with claim 3, wherein thechemical treatment is galenical presentation with an oily liquid.
 13. Amethod in accordance with claim 3, wherein said inactivating stepincludes modification of the amino acid sequence of said polypeptide,said modification being insertions, deletions or substitutions ofresidues in the amino acid sequence of said polypeptide.
 14. A method inaccordance with claim 13, wherein said inactivating step includesmodification of the amino acid sequence of said polypeptide to obtain afragment of said polypeptide.
 15. A method in accordance with claim 1,wherein said inactivating step comprises carboxyamidating said factor ora fragment of said factor, which fragment is from 8 to 110 amino acidresidues in length such that the carboxyamidated form thereof is capableof raising antibodies that neutralize the immunosuppressive and/orangiogenic activity of said factor.